1. Field of the Invention
The present invention relates to a method to improve the activity of engineered U7 snRNAs used in the context of RNA-based therapeutics; particularly in exon skipping, exon inclusion, and mRNA eradication strategies.
2. Description of Related Arts
Conventional gene therapy has focused largely on gene replacement in target cells. RNA-based strategies offer a series of novel therapeutic applications, including altered processing of the target pre-mRNA transcript, reprogramming of genetic defects through mRNA repair, and the targeted silencing of allele- or isoform-specific gene transcripts. Similarly, in disorders of RNA processing, such as aberrant splicing, it may be preferable to repair the endogenous splicing pattern, which could also correct multiple alternative isoforms.
Many genes use alternative splicing to generate multiple gene products. Being able to modulate the splicing pathway of a particular gene (on demand alternative splicing) has many potential applications in the field of gene therapy. For instance, the forced skipping of a precise exon might be used to inhibit gene function or to promote synthesis of an internally deleted or truncated protein, depending on whether the remaining exons are fused in- or out-of-frame. Similarly, forced inclusion of an exon, which is abnormally spliced from the final mRNA, would also be clinically relevant in many pathological inherited conditions.
Neuromuscular disease refers to a group of hereditary muscle diseases that weaken the muscles that move the human body. They include such diseases as e.g. neuromuscular dystrophies and spinal muscular atrophy. Nine diseases including Duchenne, Becker, limb girdle, congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal, and Emery-Dreifuss are always classified as neuromuscular dystrophy but there are more than 100 diseases in total with similarities to neuromuscular dystrophy. These conditions have a genetic basis, and the different genetic muscular diseases follow various inheritance patterns. The best-known type, Duchenne neuromuscular dystrophy (DMD), is a severe recessive X-linked form characterized by the absence of a 427 kDa protein denominated dystrophin. The absence of a functional dystrophin protein is due to a disruption of translation caused by nonsense or deletion mutations in the dystrophin gene, a large gene located at Xp21.2 (Muntoni et al., Lancet Neurol., 2: 731-740, 2003). A strategy for correcting DMD consists in using antisense oligonucleotides (AON) to skip some exons and thus express a truncated, yet functional, form of the protein. This technique, designated exon skipping, uses oligonucleotides complementary to the sequences involved in the splicing of the exons to skip (Wood et al., PLoS Genet., 3 (6): e109, 2007; Du & Gatti, Curr Opin Mol Ther., 11(2):116-23, 2009). In particular, the present inventors have shown that it is possible to correct DMD by exon skipping in mouse by using a modified U7 snRNA redirected to the spliceosome by replacement of the endogenous sm-binding domain with the one of the U1 snRNA, the smOPT (WO 2006/021724; Goyenvalle et al., Science, 306: 1796-1799, 2004). However, this technique has not yet been adapted for use in human patients.
Another important dystrophy is myotonic dystrophy (dystrophia myotonica, DM), of which the type 1 (DM1, also known as Steinert's disease) is the most prevalent. DM1 is a dominant inherited disease, caused by expanded CTG repeats in the 3′ untranslated region of the DM protein kinase (DMPK) gene (Gene map locus: 19q13.2-q13.3). The mutant DMPK mRNA is trapped in the nucleus and the CUG expansion alters binding of RNA-binding proteins to the molecule (Davis et al., Proc. Natl. Acad. Sci. U.S.A., 94: 7388-7393, 1997). Such an accumulation alters the regulation of alternative splicing, which subsequently leads to mis-splicing of several pre-mRNA transcripts and neuromuscular dysfunction. Strategies for phenotype rescue in DM1 have been evaluated with the use of AON targeting CUG expansions in murine DM1 models (Wheeler et al., Science, 325: 336-339, 2009; Mulders et al., Proc. Natl. Acad. Sci. U.S.A., 106: 13915-13920, 2009; Du & Gatti, Curr Opin Mol Ther., 11(2):116-23, 2009). However, the use of synthetic oligonucleotides requires repeated treatments. Indeed, there is currently no cure for or treatment specific to myotonic dystrophy.
Apart from the muscular dystrophies, another important muscular disease is Spinal Muscular Atrophy (SMA): it is the most common cause of genetically determined neonatal death. SMA is a hereditary neuromuscular disease characterized by degeneration of motor neurons, resulting in progressive muscular atrophy (wasting away) and weakness. The disorder is caused by an abnormal or missing gene known as the survival motor neuron gene, which is responsible for the production of the Survival Motor Protein (SMN), a protein essential to motor neurons. In humans, there are two copies of the SMN gene, named SMN1 and SMN2, and both mapped to the 5q12.2-q13.3 locus. Inactivation of the SMN1 gene leads to disease because the SMN2 gene cannot compensate for its absence. The reason for this is a critical C to U substitution in exon 7 of the SMN2 gene that does not change the codon but prevents its recognition by the splicing machinery. As a result of this substitution, the SMN2 gene predominantly (90% of the transcript) produces a transcript where exon 7 is skipped, leading to the production of a truncated protein and inability to compensate for SMN1 inactivation.
Current strategies for developing SMA therapeutics include identifying drugs that increase SMN2 levels, enhance residual SMN2 function, or otherwise compensate for the loss of SMN1 activity. Drugs such as butyrates, valproic acid, hydroxyurea, and riluzole (Rilutek®, Sanofi-Aventis) are or have been under clinical investigation for SMA. Although gene replacement strategies are being tested in animals (Foust et al., Nat Biotechnol., 28(3): 271-4, 2010), current treatment for SMA consists of prevention and management of the secondary effect of chronic motor unit loss. There is currently no drug known to alter the course of SMA.
There is thus still a need for new treatments of neuromuscular diseases. In particular, the inventors have developed a new modified human U7 snRNA which comprises a smOPT domain and a sequence antisense to at least a part of the target pre-mRNA. They have shown that an interaction between the antisense moiety and the U7 loop is required to obtain active U7-derived snRNPs which can be used for treating neuromuscular diseases.